In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid
and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The
highest survival rates were obtained when somatic embryos were encapsulated in calcium
alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with
daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic
embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight
basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1ºC min-1
from 25ºC to -30ºC followed by immersion in LN). Beads were kept in LN for a minimum of
1 h and then were rapidly rewarmed in a 30ºC water-bath for 2 min. Finally, encapsulated
somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing
sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol,
we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid
and triploid cytotypes. No morphological abnormalities were observed in any of the plants
regenerated from cryopreserved embryos and their genetic stability was confirmed with 10
isozyme systems and nine RAPD profiles.